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Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

Published on: July 6, 2016

Reversible fluorescence photoswitching in DNA.

Darren A Smith¹, Philipp Holliger, Cristina Flors

¹EaStChem School of Chemistry, University of Edinburgh , Joseph Black Building, The King's Buildings, West Mains Rd, Edinburgh EH9 3JJ, United Kingdom.

The Journal of Physical Chemistry. B

|August 7, 2012

Summary

Engineered DNA with high-density cyanine dye labeling ("CyDNA") exhibits reversible photoswitching. This breakthrough enables advanced fluorescence microscopy, including super-resolution imaging.

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Design and Synthesis of a Reconfigurable DNA Accordion Rack

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Published on: August 15, 2018

Area of Science:

Biophysics
Nanotechnology
Molecular Biology

Background:

High-density labeling of DNA with cyanine dyes (Cy3, Cy5) is challenging due to fluorescence quenching.
Previous studies suggested fluorescence quenching at high labeling densities in DNA.

Purpose of the Study:

To engineer reversible fluorescence photoswitching in DNA with high-density substitution.
To investigate fluorescence quenching mechanisms in densely labeled DNA.
To explore applications in advanced fluorescence microscopy.

Main Methods:

Developed a modified DNA polymerase for efficient incorporation of Cy3- and Cy5-labeled cytosine base analogues.
Utilized polymerase chain reaction for high-density DNA labeling.
Investigated fluorescence quenching using spectroscopic methods.
Engineered reversible photoswitching using the Cy3-Cy5 dye pair.

Main Results:

Created "CyDNA" with hundreds of fluorophores per DNA strand, exhibiting strong color and fluorescence.
Identified aggregate formation and resonance energy transfer as key fluorescence quenching mechanisms.
Successfully engineered CyDNA for reversible fluorescence photoswitching.
Demonstrated the utility of photoswitchable CyDNA in optical lock-in detection imaging.

Conclusions:

High-density DNA labeling with cyanine dyes is achievable and can be engineered for reversible photoswitching.
Understanding quenching mechanisms is crucial for optimizing densely labeled DNA nanomaterials.
Photoswitchable CyDNA represents a novel photoactive DNA-based nanomaterial with significant potential for super-resolution fluorescence microscopy and advanced imaging techniques.